The ability to detect increased levels of matrix metalloproteinases (MMPs) in the heart would be extremely useful for the detection of tissue degradation which occurs in many heart conditions. The composition and vulnerability of atheromatous plaque in the coronary arteries has recently been recognized as a key determinant in thrombus-mediated acute coronary events, such as unstable angina, myocardial infarction and death (Falk E, Shah P K and Fuster V; Circulation 1995; 92: 657-671). Among the many components involved in the inflammatory atheromatous plaque are macrophages which secrete the matrix metalloproteinases (Davies M J; Circulation 1996; 94: 2013-2020). The MMPs are a family of enzymes which specialize in the cleavage of the usually protease-resistant fibrillar extracellular matrix components of the heart, such as collagen. These extracellular matrix proteins confer strength to the fibrous cap of atheroma (Libby P, Circulation 1995; 91: 2844-2850).
Macrophages which accumulate in areas of inflammation such as atherosclerotic plaques release these MMPs which degrade connective tissue matrix proteins (Falk, 1995). In fact, studies have demonstrated that both the metalloproteinases and their mRNA are present in atherosclerotic plaques (Coker M L, Thomas C V, Clair M J, et al.; Am. J. Physiol. 1998; 274:H1516-1523; Dollery C M, McEwan J R, Henney A., et al.; Circ. Res. 1995; 77:863-868; Henney A., Wakeley P., Davies M., et al.; Proc Natl Acad Sci 1991; 88:8154-8158), particularly in the vulnerable regions of human atherosclerotic plaques (Galis Z, Sukhova G, Lark M. and Libby P.; J Clin Invest. 1994; 94: 2493-2503). Amongst the metalloproteinases that may be released by macrophages present at the site of human atheroma are interstitial collagenase (MMP-1), gelatinases A and B (MMP-2 and MMP-9, respectively) and stromelysin (MMP-3 Moreno P R, Falk E., Palacious I F et al.; Circulation 1994; 90: 775-778). Although all MMPs may be elevated at the site of human atheroma, it has been suggested that gelatinase B may be one of the most prevalent MMPs in the plaque because it can be expressed by virtually all activated macrophages (Brown D., Hibbs M, Kerney M., et al.; Circulation 1995; 91: 2125-2131). The MMP-9 has also been shown to be more prevalent in atherectomy material from unstable angina relative to stable angina patients (Brown, 1995).
The left ventricular extracellular matrix, containing a variety of collagens and elastin, are also proposed to participate in the maintenance of left ventricle (LV) geometry. Therefore, alterations in these extracellular components of the myocardium may influence LV function and be a marker of progressive changes associated with LV degeneration and ultimately heart failure (Coker, 1998).
In the situation of congestive heart failure (CHF), the relationship of CHF state to MMP activity in the LV remains somewhat unclear, at least in the clinical setting. In pre-clinical models of CHF, however, the functional changes in the LV have been correlated with increased MMP activity. For example, in a pig model of CHF, the decrease in LV function was observed to coincide with a marked increase in MMP-1 (˜300%), MMP-2 (˜200%), and MMP-3 (500%) (Coker, 1998). Moderate ischemia and reperfusion in a pig model has been demonstrated to selectively activate MMP-9 (Lu L, et al., Circulation, 1999, 100 Suppl. 1, I-12). Similarly in a dog model of CHF the levels of gelatinases (e.g. MMP-2 and MMP-9) were found to be elevated in severe heart failure (Armstrong P W, Moe G W, et al., Can J Cardiol 1994; 10: 214-220). The levels of MMP-2 and MT1-MMP (membrane type MMP, MMP-14) were found to be increased in biopsy samples of human myocytes from patients suffering from dilated cardiomyopathy (Bond B R, et al., Circulation, 1999, 100 Suppl. 1, I-12).
Ahrens, et al. U.S. Pat. No. 5,674,754 discloses methods for the detection of Matrix Metallo-Proteinase No. 9, using antibodies which selectively recognize pro-MMP-9 and complexes of pro-MMP-9 with tissue inhibitor of matrix metallo proteinase-1 (TIMP-1), with no substantial binding to active MMP-9. Venkatesan, et al. U.S. Pat. No. 6,172,057 discloses non-peptide inhibitors of matrix metalloproteinases (MMPs) and TNF-.alpha. converting enzyme (TACE) for the treatment of arthritis, tumor metastasis, tissue ulceration, abnormal wound healing, periodontal disease, bone disease, diabetes (insulinresistance) and HIV infection.
Pathologically, MMPs have been identified as associated with several disease states. For example, anomalous MMP-2 levels have been detected in lung cancer patients, where it was observed that serum MMP-2 levels were significantly elevated in stage IV disease and in those patients with distant metastases as compared to normal sera values (Garbisa et al., 1992, Cancer Res., 53: 4548, incorporated herein by reference.). Also, it was observed that plasma levels of MMP-9 were elevated in patients with colon and breast cancer (Zucker et al., 1993, Cancer Res. 53: 140 incorporated herein by reference).
Elevated levels of stromelysin (MMP-3) and interstitial collagenase (MMP-1) have been noted in synovial fluid derived from rheumatoid arthritis patients as compared to post-traumatic knee injury (Walakovits et al., 1992, Arth. Rheum., 35: 35) incorporated herein by reference. Increased levels of mRNA expression for collagenase type I (MMP-1) and collagenase type IV (MMP-2) have been shown to be increased in ulcerative colitis as compared to Crohn's disease and controls (Matthes et al., 1992, Gastroenterology, Abstract 661, incorporated herein by reference). Furthrmore, Anthony et al., 1992, Gastroenterology, Abstract 591, demonstrated increased immuno-histochemical expression of the gelatinase antigen in arabbit model of chronic inflammatory colitis.
It has been shown that the gelatinase MMPs are most intimately involved with the growth and spread of tumors. It is known that the level of expression of gelatinase is elevated in malignancies, and that gelatinase can degrade the basement membrane which leads to tumor metastasis. Angiogenesis, required for thegrowth of solid tumors, has also recently been shown to have a gelatinase component to its pathology. Furthermore, there is evidence to suggest that gelatinase is involved in plaque rupture associated with atherosclerosis. Other conditions mediated by MMPs are restenosis, MMP-mediated osteopenias, inflammatory diseases of the central nervous system, skin aging, tumor growth, osteoarthritis, rheumatoid arthritis, septic arthritis, corneal ulceration, abnormal wound healing,bone disease, proteinuria, aneurysmal aortic disease, degenerative cartilage loss following traumatic joint injury, demyelinating diseases of the nervous system,cirrhosis of the liver, glomerular disease of the kidney, premature rupture of fetal membranes, inflammatory bowel disease, periodontal disease, age relatedmacular degeneration, diabetic retinopathy, proliferative vitreoretinopathy, retinopathy of prematurity, ocular inflammation, keratoconus, Sjogren's syndrome, myopia, ocular tumors, ocular angiogenesis/neo-vascularization and corneal graft rejection. For recent reviews, see: (1) Recent Advances in Matrix Metalloproteinase Inhibitor Research, R. P. Beckett, A. H. Davidson, A. H. Drummond, P. Huxley and M. Whittaker, Research Focus, Vol. 1, 16-26,(1996), (2) Curr. Opin. Ther. Patents (1994) 4(1): 7-16, (3) Curr. Medicinal Chem. (1995) 2: 743-762, (4) Exp. Opin. Ther. Patents (1995) 5(2): 1087-110, (5) Exp. Opin. Ther. Patents (1995) 5(12): 1287-1196, all of which are incorporated herein by reference.
Therefore, the present imaging agents targeted to one or more MMP's would be very useful for detecting and monitoring the degree of extracellular matrix degradation in CHF, atherosclerosis and other degradative disease processes. These imaging agents, containing a ligand directed at one or more MMP's (e.g. MMP-1, MMP-2, MMP-3, MMP-9), will localize a diagnostic imaging probe to the site of pathology for the purpose of non-invasive imaging of these diseases. The imaging agent may be a MMP inhibitor linked to radioisotopes which are known to be useful for imaging by gamma scintigraphy or positron emission tomography (PET). Alternatively, the MMP targeting ligand could be bound to a single or multiple chelator moieties for attachment of one or more paramagnetic metal atoms, which would cause a local change in magnetic properties, such as relaxivity or susceptibility, at the site of tissue damage, which could then be imaged with magnetic resonance imaging systems. Alternatively, the MMP inhibitor can be bound to a phospholipid or polymer material which would be used to encapsulate/stabilize microspheres of gas which would be detectable by ultrasound imaging following localization at the site of tissue injury.
Therefore, imaging agents based on MMP inhibitors would be extremely useful in the detection, staging and monitoring of cardiovascular diseases such as atherosclerosis (especially unstable arterial plaque) and various cardiomyopathies including congestive heart failure. Compounds of the present invention, which localize in areas of MMP activity in the heart, will allow detection and localization of these cardiac diseases which are associated with altered MMP levels relative to normal myocardial tissue.
These imaging agents, whether for gamma scintigraphy, positron emission tomography, MRI, ultrasound or x-ray image enhancement, have utility to detect and monitor changes in cardiovascular diseases over time. Since the degree of overexpression of MMPs is related to the degradation of cardiac or vascular tissue (Rohde L E, Aikawa M, Cheng G C, et al., JACC 1999; 33: 835-842) it is possible to assess the severity and current activity of cardiovascular disease lesions (i.e. plaques) by quantitating the degree of localization of these imaging agents at the diseased sites of interest. Moreover, with these imaging agents it is possible to monitor changes in MMP activity associated with the institution of pharmaceutical therapies which slow the progression or cause a reversal of atheroschlerotic changes in the vascular system or a reversal of myocardial degradation associated with congestive heart failure.
Therefore, it can be appreciated that the imaging of MMPs in the heart would be generally useful for the detection, localization and monitoring the progression/regression of a variety of cardiac diseases which are associated with alterations in the MMP content of cardiac tissues.